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2 protocols using anti p perk

1

Western Blot Analysis of UPR Proteins

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Protein was extracted from the cultured cells using RIPA buffer. A total of 30 μg proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in TBST containing 5% skimmed milk, and incubated with the primary antibodies including: anti-GRP78 (1:1000; Sigma Aldrich), anti-PERK (1:1000; Sigma Aldrich), anti-p-PERK (1:500; Sigma Aldrich), anti-eIF2α (1:1000; Abcam, Cambridge, UK), anti-p-eIF2α (1:500; Abcam), anti-ATF4 (1:1000; Sigma Aldrich), anti-CHOP (1:500; Sigma Aldrich), anti-SIRT1 (1:1000; CST, Beverly, USA), anti-Bcl-2 (1:1000; Proteintech, Wuhan, China), Bax (1:1000; Proteintech), anti-Mcl-1(1:1000; Sigma), anti-caspase 3 (1:1000; CST), anti-caspase 9 (1:1000; CST), anti-cleaved caspase 3 (1:500; Sigma Aldrich), and anti-β-actin (1:1000; Origene, Rockville, USA), followed by incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. Finally, chemiluminescent substrate (Beyotime) was used for signal visualization and detection. The density of each band was normalized to its respective loading control (β-actin). The immunoblots were quantified by densitometric analysis.
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2

Immunoblotting and Quantification of ppErk

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For immunoblotting, cells were lysed in RIPA buffer and lysates were separated on polyacrylamide gels before transfer to nitrocellulose membranes. Antibodies used were anti-ppErk (Sigma, M9692) and anti-Erk1/2 (Millipore, 06-182) at 1:500 dilution. Detection was performed using fluorescently labeled secondary antibodies at 0.1 μg/ml (LI-COR) and scanning in a LI-COR Odyssey system. Intensities of bands were quantified in ImageStudio (LI-COR).
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